Plant oils are an important source of dietary fat for humans, representing about 25% of caloric intake in developed countries (Broun et al., 1999). The current world production of plant oils is about 110 million tones per year, of which 86% is used for human consumption. Almost all of these oils are obtained from oilseed crops such as soybean, canola, sunflower, cottonseed and groundnut, or plantation trees such as palm, olive and coconut (Gunstone, 2001; Oil World Annual, 2004). The growing scientific understanding and community recognition of the impact of the individual fatty acid components of food oils on various aspects of human health is motivating the development of modified vegetable oils that have improved nutritional value while retaining the required functionality for various food applications. These modifications require knowledge about the metabolic pathways for plant fatty acid synthesis and genes encoding the enzymes for these pathways (Liu et al., 2002a; Thelen and Ohlrogge, 2002).
Considerable attention is being given to the nutritional impact of various fats and oils, in particular the influence of the constituents of fats and oils on cardiovascular disease, cancer and various inflammatory conditions. High levels of cholesterol and saturated fatty acids in the diet are thought to increase the risk of heart disease and this has led to nutritional advice to reduce the consumption of cholesterol-rich saturated animal fats in favour of cholesterol-free unsaturated plant oils (Liu et al., 2002a). While dietary intake of cholesterol present in animal fats can significantly increase the levels of total cholesterol in the blood, it has also been found that the fatty acids that comprise the fats and oils can themselves have significant effects on blood serum cholesterol levels. Of particular interest is the effect of dietary fatty acids on the undesirable low density lipoprotein (LDL) and desirable high density lipoprotein (HDL) forms of cholesterol in the blood. In general, saturated fatty acids, particularly myristic acid (14:0) and palmitic acid (16:0), the principal saturates present in plant oils, have the undesirable property of raising serum LDL-cholesterol levels and consequently increasing the risk of cardiovascular disease (Zock at al., 1994; Hu et al., 1997). However, it has become well established that stearic acid (18:0), the other main saturate present in plant oils, does not raise LDL-cholesterol, and may actually lower total cholesterol (Bonanome and Grundy, 1988; Dougherty at al., 1995). Steric acid is therefore generally considered to be at least neutral with respect to risk of cardiovascular disease (Tholstrup, et al., 1994). On the other hand, unsaturated fatty acids, such as the monounsaturate oleic acid (18:1) and the polyunsaturates linoleic acid (18:2) and α-linolenic acid (ALA, 18:3), have the beneficial property of lowering LDL-cholesterol (Mensink and Katan, 1989; Roche and Gibney, 2000), thus reducing the risk of cardiovascular disease.
Although nutritionally desirable, highly unsaturated oils are too unstable for use in many food applications, particularly for commercial deep-frying where they are exposed to high temperatures and oxidative conditions for long periods of time. Under such conditions, the oxidative breakdown of the numerous carbon double bonds present in unsaturated oils results in the development of short-chain aldehyde, hydroperoxide and keto derivatives, imparting undesirable flavours and reducing the frying performance of the oil by raising the level of polar compounds (Chang et al., 1978; Williams at al., 1999).
Oil processors and food manufacturers have traditionally relied on hydrogenation to reduce the level of unsaturated fatty acids in oils, thereby increasing their oxidative stability in frying applications and also providing solid fats for use in margarine and shortenings. Hydrogenation is a chemical process that reduces the degree of unsaturation of oils by converting carbon double bands into carbon single bonds. Complete hydrogenation produces a fully saturated fat. However, the process of partial hydrogenation results in increased levels of both saturated fatty acids and monounsaturated fatty acids. Some of the monounsaturates formed during partial hydrogenation are in the trans isomer form (such as elaidic acid, a trans isomer of oleic acid) rather than the naturally occurring cis isomer (Sebedio et al., 1994; Fernandez San Juan, 1995). In contrast to cis-unsaturated fatty acids, trans-fatty acids are now known to be as potent as palmitic acid in raising serum LDL cholesterol levels (Mensink and Katan, 1990; Noakes and Clifton, 1998) and lowering serum HDL cholesterol (Zock and Katan, 1992), and thus contribute to increased risk of cardiovascular disease (Ascherio and Willett, 1997). As a result of increased awareness of the anti-nutritional effects of trans-fatty acids, there is now a growing trend away from the use of hydrogenated oils in the food industry, in favour of fats and oils that are both nutritionally beneficial and can provide the required functionality without hydrogenation, in particular those that are rich in either oleic acid where liquid oils are required or stearic acid where a solid or semi-solid fat is preferred.
Plant oils are composed almost entirely of triacylglycerols (TAG) molecules, which consist of three fatty acid (acyl) chains esterified to a glycerol backbone and are deposited in specialised oil body structures called oleosomes (Stymne and Stobart, 1987). These storage lipids serve as an energy source for the germinating seedling until it is able to photosynthesise. Edible plant oils in common use are generally comprised of five main fatty acids—the saturated palmitic and stearic acids, the monounsaturated oleic acid, and the polyunsaturated linoleic and α-linolenic acids. In addition to fatty acids, plant oils also contain some important minor components such as tocopherols, phytosterols, terpenes and mixed isoprenoids. These minor constituents are of increasing interest because some have been shown to exert beneficial effects on skin health, aging, eyesight and blood cholesterol or preventing breast cancer or cardiovascular disease (Theriault at al., 1999; Moghadasian and Frohlich, 1999).
Fatty Acid and TAG Synthesis in Seeds
A diagrammatic overview of the metabolic pathways for fatty acids synthesis in developing seeds is shown in FIG. 1. The initial stages of fatty acid synthesis occur in the plastid compartments of the cell, where synthesis of fatty acid carbon chains is initiated with a C2 molecule and extended through a stepwise condensation process whereby additional C2 carbon units are donated from malonyl-ACP to the elongating acyl chains. The first step in this sequence involves acetyl-CoA condensing with malonyl-ACP and is catalysed by the β-ketoacyl synthase III (KASIII) enzyme. The subsequent condensation rounds are catalysed by β-ketoacyl synthase I (KASI) and result in the eventual formation of a saturated C16 acyl chain joined to acyl carrier protein (ACP), palmitoyl-ACP. The final elongation within the plastid is catalysed by β-ketoacyl synthase II (KASII) to form the saturated C18 acyl chain, stearoyl-ACP. When desaturation occurs, the first double bond is introduced into the Δ9 position of the C18 chain by a soluble enzyme in the plastid, stearoyl-ACP Δ9-desaturase, to yield the monounsaturated C18:1 oleoyl-ACP.
Fatty acids thus synthesised are either retained in the plastid for further modification and incorporation into plastidic lipids, or are released from their ACPs by acyl-thioesterases to produce free fatty acids which are exported into the cytosol for further modification and eventual incorporation into TAG molecules. Higher plants have been found to have at least two types of acyl-thioesterase, FatA with substrate specificity towards oleoyl-ACP, an unsaturated acyl-ACP, and FatB with preference for saturated acyl-ACPs (Jones at al., 1995; Voelker at al., 1996).
On exiting the plastids, free fatty acids become esterified to Co-enzyme A (CoA) and are then available for transfer to glycerol 3-phosphate (G-3-P) backbones to form lysophosphatidic acid (LPA), phosphatidic acid (PA) and phosphatidyl-choline (PC). Additionally, in some plants, notably the Brassica species, oleic acid esterified to CoA is able to be elongated to form eicosenoic acid (C20:1) and erucic acid (C22:1). Oleic acid esterified to PC is available for further modification before incorporation into TAG. In edible oils, the principal modifications on PC are the sequential desaturations of oleic acid to form linoleic and α-linolenic acids by the microsomal Δ12-desaturase (Fad2) and Δ15-desaturase (Fad3) enzymes respectively.
Modification of Existing Fatty Acid Biosynthetic Enzymes
Gene inactivation approaches such as post transcriptional gene silencing (PTGS) have been successfully applied to inactivate fatty acid biosynthetic genes and develop nutritionally improved plant oils in oilseed crops. For example, soybean lines with 80% oleic acid in their seed oil were created by cosuppression of the Fad2 encoded microsomal Δ12-desaturase (Kinney, 1996). This reduced the level of Δ12-desaturation and resulted in accumulation of high amounts of oleic acid. Using a similar approach, cosuppression-based silencing of the Fad2 gene was used to raise oleic acid levels in Brassica napus and B. juncea (Stoutjesdijk at al., 2000). Likewise, transgenic expression in cottonseed of a mutant allele of the Fad2 gene obtained from rapeseed was found to be able to suppress the expression of the endogenous cotton Fad2 gene and resulted in elevated oleic acid content in about half of the primary transgenic cotton lines (Chapman et al., 2001). In another variation, transgenic expression in soybean of a Fad2 gene terminated by a self-cleaving ribozyme was able to inactivate the endogenous Fad2 gene resulting in increased oleic acid levels (Buhr et al., 2002). RNAi-mediated gene silencing techniques have also been employed to develop oilseeds with nutritionally-improved fatty acid composition. In cottonseed, transgenic expression of a hairpin RNA (hpRNA) gene silencing construct targeted against ghFad2-1, a seed-specific member of cotton Fad2 gene family, resulted in the increase of oleic acid from normal levels of 15% up to 77% of total fatty acids in the oil (Liu at al., 2002b). This increase was mainly at the expense of linoleic acid which was reduced from normal levels of 60% down to as low as 4%.
Fatty Acids in Cereals
In contrast to the considerable work done on fatty acid biosynthesis and modification in oilseeds, oil modification in cereals is relatively unexplored. This is probably due to the much lower levels of oils (about 1.5-6% by weight) in cereal grains and consequently the perceived lower importance of oils from cereals in the human diet.
Rice (Oryza sativa L.) is the most important cereal crop in the developing world and is grown widely, particularly in Asia which produces about 90% of the world total. The vast majority of rice in the world is eaten as “white rice” which is essentially the endosperm of the rice grain, having been produced by milling of harvested grain to remove the outer bran layer and germ (embryo and scutellum). This is done primarily because “brown rice” does not keep well on storage, particularly under hot tropical conditions.
The oil content of cereal grains such as rice (4%) is quite low relative to oilseeds where oil can make up to 60% of the weight of the grain (Ohlrogge and Jaworski, 1997). However, lipids may still comprise up to 37% of the dry weight of the cereal embryo (Choudhury and Juliano 1980). Most of the lipid content in the rice grain is found in the outer bran layer (Resurreccion et al., 1979) but some is also present in the endosperm, at least some associated with the starch (Tables 1 and 2). The main fatty acids in rice oil are palmitic (16:0) (about 20% of total fatty acids in the TAG), oleic (18:1) (about 40%), and linoleic acids (18:2) (about 34%) (Radcliffe, et al., 1997). There is a range of levels naturally occurring in different rice cultivars, for example for oleic acid, from 37.9% to 47.5% and for linoleic (18:2), from 38.2% to 30.4% (Taira et al., 1988).
TABLE 1Typical fatty acid composition (wt % of total fatty acids)for selected fatty acids of various plant oils.Plant16:018:118:2Barley182254Soybean112351Peanut85036Canola46320Olive15759Rice bran223834
While the FatB gene has been shown to have a high affinity to catalysing the production of free palmitic acid which is subsequently converted to palmitoyl-CoA in oilseed plants and dicot plants, no information is reported on the role of FatB in rice or other cereals.
TABLE 2Fatty acid composition (wt % of total fatty acids) for selectedfatty acids of plant lipids associated with starch.Plant16:018:118:2Wheat35-446-1442-52Barley55 436Rye234135Oat402235Maize371146Maize- High amylose362038Maize- waxy362336Millet362829Rice37-489-1829-46Data adapted from: Morrison (1988).
Some of the fatty acid desaturases have been characterized in rice but not Fad2. Akagai et al, (1995) published the nucleotide sequence of a gene on rice chromosome 4 encoding stearoyl-acyl carrier protein desaturase from developing seeds. The gene product participates in the production of oleoyl ACP from stearoyl ACP. Kodama et al. (1997) reported the structure, chromosomal location and expression of Fad3 in rice.
The proportion of linolenic acid (18:3) in rice seed oil has been increased ten-fold by using soybean Fad3 expression (Anai et al., 2003). More recently, there have been reports of the production of conjugated linoleic acid in rice by introduction of a linoleate isomerase gene from bacteria (Kohno-Murase at al., 2006); conjugated linoleic acid is reported to have anti-carcinogenic activity. In a similar vein, in vitro modification of rice bran oil to incorporate capric acid, which may improve dietary lipid utilisation in some diseases, using immobilized microbial enzymes has also been reported (Jennings and Akoh, 2000). In maize, Fad2 and FA-6 desaturase genes have been sequenced and mapped to chromosomes (Mikkilinen and Rocheford, 2003). The Fad2 and Fad6 clones could not be mapped to any QTLs for oleic/linoliec acid ratios in the maize grain. There are no published reports of other Fad2 or FatB genes characterized from rice, maize or wheat.
Storage of Rice
Storage of rice for prolonged times at high temperatures impairs grain quality due to hydrolytic and oxidative deterioration of bran oil. Dehulling the outer husk during harvest to produce brown rice disturbs the outer bran layers, which allows the oil to diffuse to the outer layers. Endogenous and microbial lipases then catalyse the hydrolysis of triglycerides to free fatty acids (FFA) which are then oxidised to produce an off-flavour (Yasumatsu et al., 1966, Tsuzuki et al., 2004, Thou et al., 2002 Champagne and Grimm, 1995).
Hexanal is the major component increased in the headspace of raw and cooked brown rice stored at high temperatures (Boggs et al., 1964, Shibuya et al., 1974, Tsugita et al., 1983). However, hexanal itself is not the main cause of the ‘off’ smell; the unattractive smell and flavour of deteriorated rice is probably due to a mixture of the volatiles that are increased after storage. These include alkanals, alkenals, aromatic aldehydes, ketones, 2-pentylfuran, 4-vinylphenol and others (Tsugita et al., 1983). Nevertheless, hexanal levels during storage have been shown to be associated with the oxidation of linoleic acid (18:2) in brown rice and therefore are a good indicator of oxidative deterioration (Shin et al., 1986).
The production of hexanal from linoleic acid can be catalysed by the enzyme lipoxygenase (LOX) (St Angelo et al., 1980). Suzuki at al. (1999) identified rice varieties lacking Lox3 and found that on storage of the mutant grain at 35° C. for 8 weeks, less hexanal was formed in the headspace vapour, both for raw and cooked brown rice grain. They also found that mutant rice formed less pentanal and pentanol.
The nutrient-rich outer rice bran layer obtained through polishing the outer layers of the rice grain is an excellent food source, containing antioxidant compounds such as tocotrienols and gamma-oryzanol which is also a phytoestrogen (Rukmini and Raghuram, 1991). The bioactive compounds present in rice bran oil have been found to lower cholesterol in humans (Most et al., 2005). These bioactive components have also been shown to improve lipid profiles in rats fed a high cholesterol diet (Ha et al., 2005). Another important component found primarily in the bran is vitamin A precursors. However, these nutritional and health benefits are lost through the polishing of rice and the consumption of white rice.
There is still a need for cereal, such as rice, varieties that produce grain with an improved oil composition for health benefits, which at the same time is more stable on storage, allowing greater use of, for example, brown rice in the human diet.